Loss of TCR-beta F1 and/or EZRIN expression is associated with unfavorable prognosis in nodal peripheral T-cell lymphomas

Abstract

Nodal peripheral T-cell lymphoma (nodal PTCL) has an unfavorable prognosis, and specific pathogenic alterations have not been fully identified. The biological and clinical relevance of the expression of CD30/T-cell receptor (TCR) genes is a topic under active investigation. One-hundred and ninety-three consecutive nodal PTCLs (89 angioimmunoblastic T-cell lymphomas (AITL) and 104 PTCL-unspecified (PTCL-not otherwise specified (NOS)) cases) were analyzed for the immunohistochemical expression of 19 molecules, involving TCR/CD30 pathways and the associations with standard prognostic indices. Mutually exclusive expression was found between CD3 and TCR-beta F1 with CD30 expression. Taking all PTCL cases together, logistic regression identified a biological score (BS) including TCR molecules (TCR-beta F1 and EZRIN) that separates two subgroups of patients with a median survival of 34.57 and 5.20 months (P<0.001). Multivariate analysis identified BS and the prognostic index for PTCL (PIT) score as independent prognostic factors. This BS maintained its significance in multivariate analysis only for the PTCL-NOS subgroup of tumors. In AITL cases, only a high level of ki67 expression was related to prognosis. A BS including molecules involved in the TCR signaling pathway proved to be an independent prognostic factor of poor outcome in a multivariate analysis, specifically in PTCL-NOS patients. Nevertheless, validation in an independent series of homogeneously treated PTCL patients is required to confirm these data.

OPEN

Citation: Blood Cancer Journal (2013) 3, e111; doi:10.1038/bcj.2013.10& 2013 Macmillan Publishers Limited All rights reserved 2044-5385/13

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ORIGINAL ARTICLE

Loss of TCR-beta F1 and/or EZRIN expression is associated with unfavorable prognosis in nodal peripheral T-cell lymphomas

SM Rodrguez-Pinilla1,2, MEC Snchez1, J Rodrguez3, JF Garca4, B Snchez-Espiridin1,4, LF Lamana5, G Sosa6, JC Rivero7,J Menrguez8, IB Gmez9, FI Camacho10, PR Guillen11, CPS Ordu a12, G Rodrguez13, C Barrionuevo14, R Franco15, M Mollejo16, JF Marco17, RD de Otazu18 and MA Piris1,19

Nodal peripheral T-cell lymphoma (nodal PTCL) has an unfavorable prognosis, and specic pathogenic alterations have not been fully identied. The biological and clinical relevance of the expression of CD30/T-cell receptor (TCR) genes is a topic under active investigation. One-hundred and ninety-three consecutive nodal PTCLs (89 angioimmunoblastic T-cell lymphomas (AITL) and 104 PTCL-unspecied (PTCL-not otherwise specied (NOS)) cases) were analyzed for the immunohistochemical expression of 19 molecules, involving TCR/CD30 pathways and the associations with standard prognostic indices. Mutually exclusive expression was found between CD3 and TCR-beta F1 with CD30 expression. Taking all PTCL cases together, logistic regression identied a biological score (BS) including TCR molecules (TCR-beta F1 and EZRIN) that separates two subgroups of patients with a median survival of 34.57 and5.20 months (Po0.001). Multivariate analysis identied BS and the prognostic index for PTCL (PIT) score as independent prognostic factors. This BS maintained its signicance in multivariate analysis only for the PTCL-NOS subgroup of tumors. In AITL cases, only a high level of ki67 expression was related to prognosis. A BS including molecules involved in the TCR signaling pathway proved to be an independent prognostic factor of poor outcome in a multivariate analysis, specically in PTCL-NOS patients. Nevertheless, validation in an independent series of homogeneously treated PTCL patients is required to conrm these data.

Blood Cancer Journal (2013) 3, e111; doi:http://dx.doi.org/10.1038/bcj.2013.10

Web End =10.1038/bcj.2013.10 ; published online 19 April 2013

Keywords: T-cell lymphoma; TCR-beta F1; EZRIN; JUNB; BLIMP1

INTRODUCTIONPeripheral T-cell lymphomas (PTCLs) are neoplasms derived from mature post-thymic T-cell lymphocytes, which account for B12%

of all lymphoid neoplasms.1 They are subclassied as either predominantly nodal or extranodal on the basis of their main site of involvement. Among nodal PTCLs, angioimmunoblastic T-cell lymphoma (AITL) and PTCL-not otherwise specied (NOS) are the most frequent tumor types.1,2 Both are aggressive diseases with a p35% 5-year overall survival (OS) probability, despite aggressive chemotherapy.2

Gene-expression proling studies have revealed that PTCL-NOS is derived from activated T cells3 with AITL harboring a gene-expression signature, suggesting derivation from follicular helper T cells.4 In spite of the progress made in their histological and immunophenotypical classication, and our understanding of their molecular biology, the molecular basis of nodal PTCLs pathogenesis and aggressiveness remains elusive. Recent ndings suggest that the survival of normal and, frequently, neoplastic T cells depends upon T-cell receptor (TCR) signaling.58 The rare translocations found in PTCL seem to conrm this observation. Thus, the chromosomal translocation t(5;9)(q33;q22) that generates the interleukin-2-inducible T-cell kinase (ITK)spleen tyrosine kinase (SYK) fusion

tyrosine kinase has been identied in a subgroup of PTCL. The inducible T-cell kinaseSYK associates constitutively with lipid rafts in T cells and triggers antigen-independent phosphorylation of TCR proximal proteins, leading to activation of downstream pathways and acute cellular outcomes that correspond to regular TCR ligation, although it is known that SYK is overexpressed in more than 90% of PTCL cases.9 In addition, inhibition of syk protein tyrosine kinase (PTK) induces apoptosis and blocks proliferation in T-cell lymphoma cell lines, highlighting its biological relevance.10

On the other hand, not all PTCLs are dependent on TCR signaling, as described by de Leval et al.4 Gene-expression arrays studies subclassify PTCLS according to gene signatures associated with the expression of CD30. The CD30-positive PTCLs overexpress many enzymes with transferase, synthase and kinase activity, and genes involved in the control of transcription, such as the Jun dimerization protein, which acts as an interleukin-2 repressor. The CD30-negative PTCLs overexpress genes involved in T-cell activation, such as the CD28 receptor, the CD69 activation antigen and molecules involved in the TCR signaling pathway.4 An inverse relationship between the levels of expression of CD30 and TCR genes was conrmed by Geissinger et al.11 However, the clinical consequences of these ndings are still unexplored or controversial.4,1113

1Lymphoma Group, Molecular Pathology Programme, Spanish National Cancer Research Centre (CNIO), Madrid, Spain; 2Pathology Department, Fundacin Jimnez Daz, Madrid, Spain; 3Oncology Department, Hospital Severo Ochoa de Legans, Madrid, Spain; 4Pathology Department, MDA, Madrid, Spain; 5Pathology Department, Complejo Hospitalario de Cceres, (Hospital San Pedro de Alcntara), Cceres, Spain; 6Pathology Department, Fundacin Hospital de Alcorcn, Madrid, Spain; 7Pathology Department, Hospital de Gran Canaria Dr Negrn, Canarias, Spain; 8Pathology Department, Hospital Gregorio Marann, Madrid, Spain; 9Pathology Department, Hospital General de Segovia, Segovia, Spain;

10Pathology Department, Hospital Universitario de Getafe, Madrid, Spain; 11Pathology Department, Hospital Universitario Prncipe de Asturias, Alcal de Henares, Madrid, Spain;

12Pathology Department, Hospital Universitario Reina Sofa, Crdoba, Spain; 13Haematology Department, Hospital Universitario Reina Sofa, Crdoba, Spain; 14Pathology Department, Instituto Nacional de Enfermedades Neoplsicas (INEN), Lima, Peru; 15Pathology Department, Istituto dei Tumori Giovanni Pascale, Naples, Italy; 16Pathology Department, Hospital Virgen de la Salud, Toledo, Spain; 17Pathology Department, Hospital Arnau de Vilanova de Valencia, Valencia, Spain; 18Pathology Department, Hospital Txagorritxu, Vitoria-Gasteiz, Spain and 19Pathology Service, Hospital Universitario Marqus de Valdecilla/IFIMAV, Santander, Spain. Correspondence: Dr SM Rodrguez-Pinilla, Pathology Department, Fundacin Jimnez Daz, Avd. Reyes Catlicos, 2, 28040 Madrid, Spain.

E-mail: mailto:[email protected]

Web End [email protected] Received 25 January 2013; revised 16 February 2013; accepted 25 February 2013

CD3/TCR-beta F1/CD30 pathways in T-cell lymphoma

SM Rodrguez-Pinilla et al

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The TCR is a multimeric complex composed of two ligand-binding glycoproteins containing variable regions (ab- or gd-TCR heterodimers), which are expressed on the cell surface in association with four CD3 molecules. TCR-ab or TCR-gd recognizes the antigen, whereas CD3 molecules control assembly and signal transduction. Each of the CD3g, CD3d and CD3e subunits contain one ITAM, and CD3z contains three ITAMs.14 Upon receptor ligation, two tyrosine residues within each ITAM are rapidly phosphorylated by a member of the src-family PTK, transforming them into high-afnity ligands for Syk PTK such as ZAP-70. The coordinated actions of Src and Syk PTK initiate a cascade of signals that ultimately leads to cell proliferation, cytokine secretion and effector functions.15 However, propagation and termination of TCR signaling needs T-cell signaling molecules (for example, PKCy, Lck and ZAP-70) to form a cluster at the site of cellcell contact, generating an immunological synapse. Moreover, the distal pole complex promotes T-cell activation by sequestering proteins that inhibit events at the immunological synapse. The ERM proteins, moesin, ezrin and radixin, regulate linkage between the cytoskeleton and plasma membrane, especially in actin-containing cell-surface structures, and are central regulators of distal pole complex formation. Only moesin and ezrin are expressed in T cells, the former being the most common.16,17

Here we have examined whether the expression of different molecules related to TCR/CD3 and CD30 signaling pathways can identify different prognostic subgroups of nodal PTCLs. The expression of some selected TCR and CD30-related markers was found to be associated with prognosis. A biological score (BS) constructed from two such markers proved to be an independent prognostic factor of survival in a multivariate analysis, considering the whole cohort of patients or solely the PTCL-NOS subgroup of tumors. Validation of these data in a homogeneously treated series of PTCL could conrm the potential usefulness of these observations.

MATERIALS AND METHODSTissue samplesWe analyzed a group of 193 consecutive nodal PTCLs from a larger series of cases submitted for diagnosis or second opinion to the CNIO Pathology Laboratory between 2000 and 2008. Only AITL and PTCL-NOS cases were considered in this study. Criteria for the diagnosis were based on the 2008 WHO classication.18,19 CD30-positive cases were considered to be PTCLNOS if none of them fullled the morphological criteria for classication as anaplastic large-cell lymphomas (ALCLs), or expressed either epithelial membrane antigen or anaplastic lymphoma kinase.18,19 Moreover,

specimens from patients with CD30-positive PTCL-NOS were restricted to those that lacked all B-cell antigens, including PAX5, and that expressed at least one T-cell-associated antigen or had a TCR gene rearrangement (data not shown). The project was supervised by the Ethical Committee of the Hospital Carlos III.

Tissue microarray constructionRepresentative areas from formalin-xed, parafn-embedded lymphomas were carefully selected on Hematoxylin and eosin stain sections and two 1-mm-diameter tissue cores were obtained from each specimen. All samples belonged to the initial diagnostic specimen. The tissue cores were precisely arrayed into a new parafn block using a tissue microarray workstation (Beecher Instruments, Silver Spring, MD, USA), following methods described elsewhere.20

ImmunohistochemistryTissue microarray sections were immunohistochemically stained by the

Endvision method with a heat-induced antigen-retrieval step. Sections were immersed in boiling 10 mM sodium citrate at pH 6.5 for 2 min in a pressure cooker. A panel of 19 antibodies related to TCR and CD30 pathways was analyzed (Supplementary Table 1). Epithelial membrane antigen, anaplastic lymphoma kinase and PAX5 were only performed in CD30-positive cases during the diagnostic procedure of the cases to exclude diffuse large B-cell lymphoma, Hodgkin lymphomas or ALCL anaplastic lymphoma kinase-positive. Staining intensity and percentage of tumoral cells were taken into consideration for scoring markers. Three

groups were established: 0, 010% positive cells with low-intensity staining; 1, 1080% positive cells, irrespective of staining intensity; 2, more than 80% positive neoplastic cells with high-intensity staining (Table 1). Depending on the marker, analyzed cases included in group 1 were considered either positive or negative. A rationale for these cutoffs is included in Supplementary Table 1. Cases positive for CD3 and TCR were subdivided into cytoplasmic or membranous groups, according to their predominant pattern of expression. Reactive tonsil tissue was included as a control. The primary antibodies were omitted to provide negative controls.

Clinical dataClinical data and the standard prognostic indices2124 for this series are

shown in Table 2. All prognostic indices were assessed at the time of initial diagnosis. The detail of therapeutic regimens for all patients is shown in Supplementary Table 2.

Statistical analysisTo assess associations between categorical variables, we used the X2 contingency test with Yates correction, or Fishers exact test, as appropriate. OS was taken as the period between the date of diagnosis and the date of death from any cause or of last contact for living patients. Disease-specic OS was calculated as the period from date of diagnosis to death from the tumor. KaplanMeier survival analyses were carried out for OS and lymphoma-specic survival, using the long-rank test to examine differences between groups. A multivariate Cox regression model was also derived. Estimates were considered statistically signicant for two-tailed probabilities of Po0.05. All analyses were carried out with SPSS 12.0 (SPSS

Inc., Chicago, IL, USA).

We also constructed a biological predictor score based on the results of the Cox functional survival curves of the signicant variables. The cutoff point for subclassifying patients as being at low- or high risk of prognosis was calculated considering the optimal sensitivity and specicity of the Cox curve. The model is represented by the following equation: h(t) h0(t)x exp(bxx byy y. bzz) (http://www.whatisseries.co.uk

Web End =www.whatisseries.co.uk).

RESULTSWhole cohort of nodal PTCL cases

There were 89 AITL (43.52%) and 104 PTCL-NOS cases (53.88%), of which 47 expressed CD30 (24.35% of the total cohort). Seventeen AITL and 19 PTCL-NOS cases had low levels of CD30 expression, whereas one AITL and 10 PTCL-NOS cases presented high levels (more than 80% of the tumoral cells) of CD30 expression. These results are in accordance with those of previous reports.12 None

of the cases expressed TCRGAMMA, PAX5, epithelial membrane antigen or anaplastic lymphoma kinase, which excludes the diagnosis of gamma-delta-T-cell lymphoma, diffuse large B-cell

Table 1. Score of the proteins analyzed in this series

Antibody Total studied cases

Number/percentage of positive cases

CD3 168 162/96.4 TCR-beta F1 164 157/95.7 TCRGAMMA 160 0/0 CD30 185 47/25.4 ZAP-70 174 147/84.5 PD1 180 122/67.7 EZRIN 148 115/77.7 MOESIN 152 147/96.7 CAVEOLIN 1 173 22/12.7 FASCIN 174 25/16.1 BLIMP1 179 25/13.9 JUNB 168 21/12.5 NF-kB-P50 146 13/8.9

NF-kB-P52 144 22/15.3 NF-kB-RELB 152 12/7.9 NF-kB-P65 173 9/5.2

Ki67 191 48/25.13

Blood Cancer Journal & 2013 Macmillan Publishers Limited

Table 2. Clinical features of the series (N 193)

Whole group Frequency Percentage

Age (years) 193 99.5 p60 79 40.9 460 114 59.1

Sex 193 99.5 Male 118 61.1 Female 75 38.9

ECOG 160 82.5 p1 114 71.3 41 46 28.8

LDH 155 79.9 Normal 70 45.2 High 85 54.8

Ann Arbor stage 165 85.1 III 41 24.8 IIIIV 124 75.2

B-symptoms 161 83.0 Yes 92 57.1 No 69 42.9

Extranodal involvement 165 85.1 o2 sites 124 75.2

X2 sites 41 24.8

BM involvement 149 76.8 Negative 107 71.8 Positive 42 28.2

Platelet cell count 76 39.2 p150 109/l 22 28.9

4150 109/l 54 71.1 IPI 163 84

Low risk 24 14.7 Low-intermediate risk 54 33.1 High-intermediate risk 53 32.5 High risk 32 19.6

PIT 150 77.3 Low risk 29 19.3 Low-intermediate risk 39 26.0 High-intermediate risk 44 29.3 High risk 38 25.3

mPIT 160 82.5 Low risk 71 44.4 Intermediate risk 49 30.6 High risk 40 25.0

IPTCLP 76 39.2 Low risk 22 38.9 Low-intermediate risk 33 43.4 High-intermediate risk 18 23.7 High risk 3 3.9

First line treatment 146 75.25 CHOP or CHOP-like 124 84.93 Others 22 15.06

Response 142 73.2 CR* 77 54.2 PR** 29 20.4 P*** 36 25.4

Recurrence 138 71.1 Yes 32 23.2 No 106 76.8

State of the patient 166 85.6 Dead 97 58.4 Alive 69 41.6

Abbreviations: BM bone marrow; CHOP combination chemotherapy

with cyclophosphamide, doxorubicin, vincristine and prednisone; ECOG performance status (Eastern Cooperative Oncology Group);

IPI international Prognostic Index; IPTCLP international peripheral

T-cell lymphoma project score; mPIT modied prognostic Index for

T-cell lymphoma; LDH serum lactate dehydrogenase; PIT prognostic

Index for T-cel lymphoma. *CR, complete response; **PR, partial response; ***P, progression.

CD3/TCR-beta F1/CD30 pathways in T-cell lymphoma SM Rodrguez-Pinilla et al

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lymphoma, Hodgkin lymphoma and ALCL,12,18,19 which are tumor

types with their own specic molecular pathogenesis.

Figure 1 shows some of the immunohistochemical characteristics of this series. Associations between the proteins analyzed in the whole series of nodal PTCL cases are shown in Supplementary Table 3A. Although P-values changed, correlations between different makers were alike, independently of the cutoff chosen for CD30 (data not shown). In general, taking all the data together, CD3 and TCR-beta F1 expression were directly correlated with each other and inversely correlated with the presence of CD30. We also found that TCR-beta F1-negative cases had cytoplasmic CD3 expression and vice versa. In addition, CD30-negative cases expressed both TCR-beta F1 and CD3 in the membrane, whereas CD30-positive cases expressed these two markers in the cytoplasm (data not shown).

Cases expressing CD3/TCR-beta F1 in the membrane expressed EZRIN. Moreover, ZAP-70 expression was associated with the presence of TCR-beta F1, whereas PD1 and MOESIN expression was associated with the presence of CD3. No signicant association was found between EZRIN and MOESIN expression.

Conversely, CD30-positive cases tended to express FASCIN, BLIMP1 and JUNB. In addition, they showed nuclear expression of p50, p52 and RelB nuclear factor kappa B (NF-kB) subunits, whereas

CD3/TCR-beta F1-positive cases more frequently expressed p65. Moreover, JUNB-positive cases showed nuclear staining of p50, p52 and RELB, whereas BLIMP1 was only signicantly associated with the presence of RELB and p52.

Caveolin-1 was not associated with the expression of CD3, TCR-beta F1 or with other cytoskeletal proteins.

Cases with a high proliferation index tended to express CD30, JUNB, BLIMP1, FASCIN and Caveolin-1, and to have nuclear expression of some NF-kB subunits, whereas those with a low proliferation index expressed CD3 and TCR-beta F1 in the membrane, as well as MOESIN.

After a mean follow-up of 23.44 months (ranging from 0 to 150 months), 75 of 162 (46.30%) nodal PTCL patients had died, and 66 of 150 (44%) patients had died because of the tumor. The overall cumulative probability of survival at 5 years was 44.4% and the tumor-specic survival probability was 51.3% (Supplementary Figure 1).

Taking all the cases together, high IPI, PIT, mPIT and IPTCLP were correlated with poor outcome in the univariate analysis (Figure 2 and Supplementary Table 4). The Cox multivariate analysis identied PIT as an independent prognostic factor of disease-specic OS (Supplementary Table 5). The expression of JUNB and BLIMP1, as well as TCR-beta F1 and EZRIN loss were signicantly associated with shorter survival in the univariate analysis (Supplementary Table 4 and6), although only TCR-beta F1 and EZRIN were identied as independent prognostic factors in the multivariate analysis (Figure 3 and Supplementary Table 5). A BS combined these two biological variables according to the equation: h(t) h0(t)x

exp(( 1.054)TCR-beta F1 ( 0.430) EZRIN) was constructed. This

was able to identify patients who were at low and high risk of dying from their disease. The low-risk group contained 63 patients (59.4%, median survival 34.57 months; range 1150 months) and there were 43 cases in the high-risk group (40.6%, median survival 5.20 months; range 114 months). The BS was highly correlated with patient outcome (Po0.001) and maintained its independent prognostic value in the Cox multivariate analysis after including PIT (Figure 4 a and Supplementary Table 5). The entire set of markers was available for 96 patients, since individual data being missing for some cases. The analysis of this smaller group of 96 cases yielded the same results (data not shown).

Analyzing data based on histological subgroups of tumors (AITL vs PTCL-NOS)

When analyzing the expression of the studied proteins according to histological diagnosis (AITL vs PTCL-NOS), we found that AITL

& 2013 Macmillan Publishers Limited Blood Cancer Journal

CD3/TCR-beta F1/CD30 pathways in T-cell lymphoma

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Figure 1. Representative pictures of nodal PTCL expressing CD3 (a), TCR-beta F1 (b) and EZRIN (c). Nodal PTCL cases negative for CD3 (d), TCR-beta F1 (e) and EZRIN (f). Nodal PTCL expressing low levels of CD30 (g). Nodal PTCL cases positive for JUNB (h) and BLIMP1 (i). A representative AITL case expressing both PD1 (j) and Caveolin-1 (k). Nodal PTCL case showing a high level of KI67 expression (l).

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Figure 2. Univariate KaplanMeier survival curves of signicant prognostic clinical indices in the present series of nodal PTCL cases.

Blood Cancer Journal & 2013 Macmillan Publishers Limited

CD3/TCR-beta F1/CD30 pathways in T-cell lymphoma SM Rodrguez-Pinilla et al

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Figure 3. Univariate KaplanMeier survival curves of signicant biological markers.

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Figure 4. (a) Univariate KaplanMeier survival curves of the BS created on the basis of the combination of biological variables that were signicant in the Cox multivariate analysis (EZRIN and TCR-beta F1) in nodal PTCL patients. (b) Univariate KaplanMeier survival curves of the BS created on the basis of the combination of biological variables that were signicant in the Cox multivariate analysis (EZRIN and TCR-beta F1) in PTCL-NOS patients.

cases were distinguished by the expression of PD1 (Po0.001) and EZRIN (P 0.010), whereas PTCL-NOS cases displayed increased

expression of JUNB (P 0.010), BLIMP1 (P 0.017), RelB

(P 0.056), p52 (0.017) and CD30 (0.029).

The relationship between most of the proteins analyzed here showed the same association as found in the entire set of patients (Supplementary Table 3B).

Patients groups were not signicantly different in terms of age of onset of the disease (P 0.106), sex (P 0.658), performance

status (P 0.205), stage (P 0.356), or low- or high-risk groups

according to various clinical prognostic indices (P 0.345; 0.181;

0.417 and 0.871 for IPI, PIT, m PIT and IPTCLP, respectively). The OS of the two groups was not signicantly different either (P 0.259;

data not shown).

The BS was analyzed for the AITL and PTCL-NOS cases separately. Interestingly, we found that the previously dened BS was only applicable to PTCL-NOS.

In the group of AITL cases, univariate analysis showed high PIT (P 0.001), mPIT (P 0.009); data not shown) and high Ki67

(P 0.022) to be indicators of poor outcome (Supplementary

Figure 3 and Supplementary Table 6). Only the PIT prognostic index maintained its prognostic value when including mPIT, KI67 and PIT in the Cox multivariate study (Supplementary Table 7).

Conversely, high scores of IPI, PIT, mPIT and IPTCLP continued to be associated with poor outcome in the univariate analysis in the PTCL-NOS tumor group (Po0.01 in all comparisons)

(Supplementary Figure 2). IPI and IPTCLP both proved to be independent prognostic factors of poor outcome in the Cox multivariate analysis (P 0.043 and P 0.029, respectively)

(Supplementary Table 7). In this group, the presence of BLIMP1 (P 0.025), and the absence of both TCR-beta F1 (Po0.001) and

EZRIN (P 0.026) were correlated with poor survival in the

univariate analysis, but only TCR-beta F1 and EZRIN maintained their signicance after multivariate analysis (Supplementary

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Table 7 and Supplementary Figure 3). A similar BS, again combining these two variables, was created. Using this BS, all high-risk patients (32 cases) died of disease before 20 months of follow-up, whereas most low-risk patients (22 cases) were alive at that time after 20 months of following up (Figure 4b). This BS also maintained its independent prognostic value in the Cox multivariate analysis after including mPIT and IPTCLP (Supplementary Table 7).

DISCUSSIONWe have investigated the expression of TCR and NF-kB-associated molecules in a large series of PTCLs. The results of the study seem to conrm that PTCL-NOS and AITL have specic diagnostic and prognostic features that justify their separate recognition. Thus, AITL more frequently expresses follicular helper T-cell markers (TFH markers), such as PD1, whereas PTCL-NOS expresses the highest levels of JUNB, BLIMP1, RelB, p52 and CD30.

The rst objective of the study was to investigate whether the expression of these molecules was hazardous, or if associations between distinct sets of molecules could contribute to a better understanding of PTCL pathogenesis. In general, nodal PTCL cases express either CD3/TCR-beta F1 or CD30. These ndings are in accordance with those of previous reports, implying that there are different subgroups of tumors whose neoplastic cells appear to use diverse intracellular machinery genes to maintain cell survival and proliferation.4,1113 Essentially, TCR-beta F1/CD3-positive tumors resembled normal T-cell lymphocytes as they expressed ZAP-70, EZRIN, MOESIN and PD1, whereas the expression of CD30 was correlated with the presence of FASCIN, JUNB and BLIMP1. Tumoral cells expressing TCR-beta F1 also expressed CD3 in their membranes, as it has been shown that surface expression of the TCR complex requires a fully assembled set of the complex subunits.25 Absence of surface CD3 (with presence of cytoplasmic CD3 expression) and TCR expression have been described in both systemic and cutaneous ALCL cases,1113 as well

as in enteropathy-associated T-cell lymphomas,26 a subgroup of tumors characterized by CD30 expression.4,1113 EZRIN is a protein

that serves as a scaffold facilitating efcient signal transduction on the cytoplasmic face of the plasma membrane, and is a negative regulator of apoptosis.27 Moreover, it is lost in acute leukemia cell lines that are resistant to antimicrotubule agents (vincristine).28

Caveolae are lipid microdomains or rafts with a high sphingolipid and cholesterol content that are thought to be the platforms for the TCR/CD3 signaling complex.29 Gene-expression arrays have revealed that Caveolin-1 is upregulated in the neoplastic cells of adult T-cell leukemia/lymphoma patients,30 and induces T-cell proliferation and NF-kB activation in a CD3/TCR-dependent manner.31 FASCIN is another actin-binding protein mainly involved in the organization of actin-based structures that function in cell adhesion and migration,32 but is little expressed, if at all, in T cells.33 In our series, FASCIN was correlated with the presence of CD30 and the loss of CD3, consistent with ndings of previous reports.34,35 Loss of EZRIN and gain of FASCIN have both

been related to epithelialmesenchymal transition, which is involved in invasiveness and metastasis.36,37 Moreover, the hedgehog signaling pathway has been found to be active in epithelialmesenchymal transition,38,39 as well as in ALCL and

some aggressive diffuse large B-cell lymphomas.40,41

The second purpose of the study was to investigate whether the expression of these TCR and NF-kB genes could provide prognostic markers. Thus, we have identied a BS based on the loss of TCR-beta F1 and EZRIN that distinguishes two prognostic groups of nodal PTCLs. This signature was exclusively maintained in the PTCL-NOS subgroup of tumors when cases were divided according to the WHO classication criteria into PTCL-NOS and AITL. In addition, this BS was independent of classical prognostic indices after multivariate analysis.

Using these thresholds, neither CD30 nor NF-kB subunit expression was signicantly associated with changes in survival probability.

In normal T cells, signaling through TCR or CD30 leads to the activation of the NF-kB pathway through a variety of intermediate molecules.4244 However, it is not known whether this activation results in the expression of the same set of pro-survival genes.45,46

In this series, NF-kB transcription factors were expressed in the nucleus of the neoplastic cells in both CD30-positive and CD30-negative nodal PTCLs. Although CD30-positive cases expressed nuclear p50, p52 and RelB NF-kB subunits, CD3/TCR-beta F1-positive cases expressed p65. In contrast to what has been found in gene-expression array studies,47 there was no association between the expression of any of the NF-kB subunits and prognosis. Discrepancies between these studies could be because of inherent differences in the techniques used.

BLIMP1 and JUNB are two NF-kB genes that are important in T-cell differentiation.44 BLIMP1 (also called PRDI-BF1 in humans) is encoded by the Prdm1 gene and is a critical transcription factor in peripheral T cells.48 Using an antibody that recognizes both PRDM1 isoforms, we noted that BLIMP1 expression was related to the PTCL-NOS subgroup of tumors, the activation of the alternative NF-kB pathway (RELB and p52) and to a poor outcome in the univariate analysis, consistent with results of previous studies of PTCL that used semiquantitative reverse transcriptase PCR.49,50

Interestingly, bortezomib (a proteasome inhibitor) has been shown to downregulate PRDM1b through NF-kB inactivation, overcoming the chemotherapy resistance in HUT-78 cell lines.51 A clinical trial using bortezomib plus CHOP in advanced aggressive PTCL yielded a complete remission rate of 61.5%.51,52 These data suggest that

BLIMP1 expression in nodal PTCLs could be a surrogate marker of prognosis and a therapeutic target.

JUNB is an activator member of the AP-1 protein transcription family.53 Its overexpression and amplication are both known to be associated with neoplastic transformation54 in CD30-positive lymphomas through an NF-kB-dependent mechanism.44 In our

series, JUNB was also associated with the presence of CD30, absence of CD3 and activation of the NF-kB pathway. Moreover, as in normal T lymphocytes,55 JUNB expression was correlated with BLIMP1 and associated with the PTCL-NOS subgroup of tumors in our series. Moreover, we also found JUNB expression to be a negative prognostic factor, our results being consistent with those of previous studies of mycosis fungoides.56

Taking these data together suggests that there are different means by which the classic and alternative NF-kB pathways are activated in distinct subgroups of nodal PTCLs, and that these warrant further investigation.

In the present series including all cases, KI67 expression by itself was not correlated with prognosis, but was stronger in the CD30-positive subgroup of tumors. These data are in accordance with the ndings of Cuadros et al.57 and Gutirrez-Garca, et al.23 and in contrast to those of Went et al.21 Surprisingly, it proved to be a predictor of poor outcome in the univariate statistical analyses for the AITL subgroup of patients. Cuadros et al.57 identied a poor prognostic signature related to proliferation using gene-expression arrays that did not differ between AITL and PTCL-NOS, although they did not study the AITL cases alone.

Tjon et al.26 suggested that the downregulation of the TCR by T-cell neoplastic cells might be a way of escaping immune-regulatory processes. Moreover, the lack of representative cell lines of most of these types of tumors and of engraftments of human tissue in immunocompromised mice, suggests that the tumoral cell depends strongly on its microenvironment.58 Here we showed that the loss of TCR-beta F1 is associated with CD30 expression, substitution of normal ezrin cytoskeletal proteins by fascin and the activation of oncogenes such as BLIMP1 and JUNB, which confer a more aggressive behavior on these tumors. In contrast with B-cell neoplasms, there is no clear proof of the

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existence of a normal counterpart for each type of T-cell lymphoma, and the ndings described here could be interpreted as evidence of the existence of tumor-specic signatures, reecting individual molecular events that characterize the neoplastic transformation of T cells.

In conclusion, we conrmed the mutually exclusive expression of TCR and CD30 molecules in PTCL, and identied some specic markers that may be used to predict survival independently of the clinical variables, specically among the PTCL-NOS tumor subgroup. Nevertheless, these patients were heterogeneously treated and immunohistochemical cutoff values were manually identied. The results would require conrmation in an independent series of homogeneously treated PTCL patients.

CONFLICT OF INTEREST

The authors declare no conict of interest.

ACKNOWLEDGEMENTS

This study was supported by grants from the Ministerio de Ciencia e Innovacin, Spain (RETICC, SAF2008-03871) and the Spanish Association against Cancer (AECC). We thank the CNIO Tumour Bank, especially Laura Cereceda and Maria Jess Artiga. We are also grateful to CI Gmez, C Gonzalez and A Gutierrez for their help with the database and the statistical analysis.

DISCLAIMERThe submitted material is original, has not been previously reported and is not under consideration for publication elsewhere. All the authors have reviewed the manuscript, agree with its content, and approve of its submission.

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